Urban waters play a crucial role in biodiversity conservation and the provision of ecosystem services. However, the drivers of changes in these habitats remain poorly understood. This study investigates the determinants of microbial community dynamics along a 0.14 km heatwave-impacted hydrological gradient formed by three connected urban ponds in Shenzhen, southern China. The experimentation focused on extremely sensitive microorganisms, testate amoebae, using 30 water samples representing water columns, 30 sediment samples representing beds, and 27 microspatial factors that capture weather conditions (WC), air quality (AQ), and water quality (WQ). Both environmental conditions and testate amoeba communities varied significantly along the heatwave-impacted hydrological gradient. A total of 39 species were recorded, marking a net increase of 5 species relative to the pre-heatwave period (June–July 2020). The mean species richness was 9 ± 1 in water and 16 ± 1 in sediment. The mean absolute abundance reached 30 ± 5 ind. mL⁻¹ in water and 2500 ± 430 ind. mL⁻¹ in sediment. Water depth and pH emerged as the most influential factors, with water depth explaining 12.4% of the total variation in testate amoeba species in water and pH accounting for 21.8% of the total variation in sediment. The variation partitioning analysis (VPA) revealed that WC, AQ, and WQ substantially influenced testate amoeba communities in sediment, accounting for 11%, 20%, and 15.5%, respectively, based on the pure percentage of community variation. Mechanistically, community changes were driven by warming-induced physiological changes, toxicity-induced stress, and hypoxia-induced stress. The neutral model explained 78.8% in water and 69.3% in sediment. Standardized effect sizes (SES) for the checkerboard score (C-score) in the null model were 0.732 (21.2%) in water and 4.437 (30.7%) in sediment, indicating that stochastic processes have a stronger influence on testate amoeba community composition than deterministic ones. These results suggest that determinants of testate amoeba community dynamics in urban waters involve both predictable factors, such as heatwaves, air pollution, and hydrological gradient (deterministic processes), and unpredictable factors, such as dispersal limitation, ecological drift, and homogenizing dispersal (stochastic processes). © 2025 Elsevier B.V., All rights reserved.